This is an ongoing comprehensive program for study at the molecular level of composition and mechanistic details of mitochondrial electron transport; energy conservation, transduction and transfer; ATP synthesis and hydrolysis. Five enzyme complexes have been isolated: I, II, III and IV are segments of the respiratory chain and capable of total respiratory activity reconstitution; V catalyzes oligomycin- and uncoupler-sensitive ATP-Pi exchange, and contains exclusively the mitochondrial uncoupler-binding site as determined by equilibrium binding and photoaffinity labeling studies using radioactive 2-azido-4-nitrophenol. The objectives of this program are (a) in complex I: to study mechanisms of NADH and NADPH oxidation and ubiquinone (Q) reduction, isolate and purify the iron-sulfur proteins bearing centers 1, 2, 3, 4, study the site 1 mechanisms of energy conservation and transfer; (b) in complex II: to study mechanism of Q reduction, roles of cytochrome b557.5 and the low-potential iron-sulfur center S-2, purify b557.5; (c) in complex III: to purify cytochromes b562 and b566, study their regulatory properties, characteirize chromophore-558, study the site 2 mechanisms of energy conservation and transfer; (d) in complex V: to study mechanisms of ATP-Pi exchange, ATP hydrolysis, uncoupling, energy conservation and transfer, components required for these functions, resolution of V into its component polypeptides and reconsitution into active ATP-Pi exchange complex, proton translocation properties of Complex V and whether vesicle formation is required for ATP-Pi exchange, reconstitution of V with I, III and IV for energy transfer and oxidative phosphorylation.